This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphorfructokinase and not generally operative in animals or encoded by the plastic DNA, to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors. A cDNA sequence that encodes chorismate synthase expressed at an early state of Apicomplexan development, is disclosed and may be altered to produce a xe2x80x9cknockoutxe2x80x9dorganism useful in vaccine production.
Apicomplexan parasites cause the serious diseases malaria, toxoplasmosis, sryptosporidiosis, and eimeriosis. Malaria kills more than 2 million children each year. Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss of life, sight, hearing, cognitive and motor function in congenitally infected infants, and considerable morbidity and mortality in patients immunocompromised by cancer, transplantation, autoimmune disease and their attendant therapies. Cryptosporidiosis is an untreatable cause of diarrhea in AIDS patients and a cause of epidemics of gastrointestinal disease in immunocompetent hosts. Eimeria infections of poultry lead to billions of dollars in losses to agricultural industries each year. Other Apicomplexan infections, such as babesiosis, also cause substantial morbidity and mortality. Although there are some methods for diagnosis and treatment of Apicomplexan caused diseases, some of these treatments are ineffective and often toxic to the subject being treated.
The tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs, agglutination of parasites or parasite components to detect antibodies to parasites, or polymerase chain reaction (PCR) to detect a parasite gene. Most of the assays utilize whole organisms or extracts of whole organisms rather than recombinant proteins or purified parasite components. In many instances, the available assays have limited ability to differentiate whether an infection was acquired remotely or recently, and are limited in their capacity to diagnose infection at the outpatient or field setting.
The primary antimicrobial agents used to treat toxoplasmosis are pyrimethamine (a DHFR inhibitor) and sulfadiazine (a PABA antagonist). The use of pyrimethamine is limited by bone marrow toxicity which can be partially corrected by the concomitant administration of folinic acid. T. gondii cannot utilize folinic acid but mammalian cells can. Another problem is that pyrimethamine is potentially teratogenic in the first trimester of pregnancy. The use of sulfonamides is limited by allergy, gastrointestinal intolerance, kidney stone formation and Stevens-Johnson syndrome.
There are a small number of antimicrobial agents utilized less frequently to treat toxoplasmosis. These include clindamycin, spiramycin, azithromycin, clarithromycin and atovaquone. Usefulness of these medicines for treatment of toxoplasmosis is limited by toxicities including allergy and antibiotic-associated diarrhea, (especially Closteidium difficile toxin associated colitis with clindamycin use). Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clarithromycin also limits use of these antimicrobial agents. Atovaquone treatment of toxoplasmosis may be associated with lack of efficacy and/or recrudescent disease. There are no medicines known to eradicate the latent, bradyzoite stage of T. gondii, which is very important in the pathogenesis of toxoplasmosis in immunocompromised individuals or those with recurrent eye disease.
Medicines used to treat malaria include quinine, sulfate, pyrimethamine, sulfadoxine, tetracycline, clindamycin, chloroquine, mefloquine, halofantrine, quinidine gluconate, quinidine dihydrochloride, quinine, primaquine and proguanil. Emergence of resistance to these medicines and treatment failures due to resistant parasites pose major problems in the care of patients with malaria. Toxicities of mefloquine include nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and seizures. Mefloquine is teratogenic in animals. With halofantrene treatment, there is consistent, dose-related lengthening of the PR and Qt intervals in the electrocardiogram. Halofantrene has caused first degree heart block. It cannot be used for patients with cardiac conduction defects. Quinidine gluconate or dihydrochloride also can be hazardous. Parenteral quinine may lead to serve hypoglycemia. Primaquine can cause hemolytic anemia, especially in patients whose red blood cells are deficient in glucose 6-phosphate dehydrogenase. Unfortunately, there are no medicines known to be effective in the treatment of cryptosporidiosis.
To more effectively treat Apicomplexan infections, there is an urgent need for discovery and development of new antimicrobial agents which are less toxic than those currently available, have novel modes of action to treat drug resistant parasites that have been selected by exposure to existing medicines, and which are effective against presently untreatable parasite life cycle stages (e.g., Toxoplasma gondii bradyzoites) and presently untreatable Apicomplexan parasites (e.g., Cryptosporidium parvum). Improved diagnostic reagents and vaccines to prevent these infections are also needed.
Information available on Apicomplexan parasites has not yet provided keys to solutions to health problems associated with the parasites. Analogies to other organisms could provide valuable insights into the operations of the parasite. There are reports of Apicomplexan parasites having plastids, as well as the nuclear encoded proteins, tubulin, calmodulin, PPi phosphofructokinase and enolase, which are reported to be similar in part to, or homologous with, counterparts in plant-like, lower life forms and higher plants. There are reports of a plastid genome and components of a protein synthetic system in a plastid-like organelle of Apicomplexans. Plasmodium and T. gondii plastid DNA sequences were reported to have homologies to algal plastid DNA sequences. The plastid membrane of T. gondii was reported to be composed of multiple membranes that appear morphologically similar to those of plant/algal chloroplasts, except for the presence of two additional membranes in the T. gondii plastid, suggesting that it may have been an ancient algal endosymbiont. Some of these Apicomplexan proteins such as tubulin, calmodulin and enolase with certain plant-like features also are found in animals, and therefore may appear in the host as well as the parasite. A homologue to a gene, psbA encoding a plant protein important for photosynthesis, also was said to be present in Apicomplexans.
Certain herbicides have been reported to inhibit the growth of Apicomplexans. The herbicides which affect growth of Apicomplexans are known to affect plant microtubules or a plant photosynthetic protein. In addition, a compound, salicylhydroxamic acid, (SHAM), had been found to inhibit Plasmodium falciparum (malaria) and Babesia microti. 
Techniques of medicinal chemistry and rational drug design are developed sufficiently to optimize rational construction of medicines and their delivery to sites where Apicomplexan infections occur, but such strategies have not yet resulted in medicines effective against Apicomplexans. Rational development of antimicrobial agents has been based on modified or alternative substrate competition, product competition, change in enzyme secondary structure, and direct interference with enzyme transport, or active site. Antisense, ribozymes, catalytic antibodies, disruption of cellular processes using targeting sequences, and conjugation of cell molecules to toxic molecules are newly discovered strategies employed to interrupt cellular functions and can be utilized to rationally develop novel antimicrobial compounds, but they have not yet been utilized to design medicines effective against Apicomplexans. Large scale screening of available compounds with recombinant enzymes is used to identify potentially effective anti-microbial agents.
Reagents to diagnose Apicomplexan parasite infections have been developed targeting components of Apicomplexans or immune responses to the parasites, using ELISA, western blot, and PCR technologies, but improved diagnostic reagents, especially those that establish duration of infection or that can be used in outpatient settings are needed to diagnose Apicomplexan infections. No vaccines to prevent Apicomplexan infections are available for humans and only a live vaccine prepared for prevention of toxoplasmosis in sheep is available for livestock.
To summarize, Apicomplexan parasites cause substantial morbidity and mortality, and treatments against the parasites are suboptimal or non-existent. Improved antimicrobial compounds that attack Apicomplexan parasites are needed. Because the diseases Apicomplexan parasites cause in some instances are due to recrudescence of latent parasites, an especially pressing clinical problem is that there are no effective antimicrobial agents effective for treatment of these latent parasite life cycle stages, especially in sequestered sites such as the brain or eye. New approaches and drug targets are required. Better in vitro and in vivo assays for candidate compounds are also needed. Better diagnostic and therapeutic methods, reagents and vaccines to prevent these infections are needed.
This invention relates uses of components of plant-like metabolic pathways (not usually associated with animals, not encoded in the plastid genome, and not including psbA or PPi phosphofructokinase) to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences, to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors. Attenuation of live parasites through disruption of any of these components or the components themselves provide vaccines protective against Apicomplexans.
Transit peptides are used to identify other proteins and their organelle targeting sequences that enter and exit from unique Apicomplexan organelles. The identified components are potential for production of medicines, reagents and assays, and vaccines. The protein which includes the transit peptide is not necessarily an enzyme in a biochemical pathway.
The methods and compositions of the present invention arise from the inventors"" discovery that metabolic pathways, and targeting signals similar to those found in plants and algae, especially, but not exclusively those encoded within the nucleus, are present in Apicomplexan parasites. These plant-like pathways in Apicomplexan parasites are targetable by inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or killing Apicomplexan parasites in vitro and/or in vivo.
The present invention includes new methods and compositions to treat, diagnose and prevent human and veterinary disease due to Apicomplexan infections.
The invention is based on applications and manipulations of components of algal and higher plant-like metabolic pathways discovered in Apicomplexan parasites. xe2x80x9cPlant-likexe2x80x9d means that products of the pathways, enzymes and nucleotides sequences encoding enzymes in the pathways, are homologous or similar to products, enzymes and nucleotide sequences known in plants, wherein plants include algae. As used herein, xe2x80x9cplant-likexe2x80x9d excludes metabolic pathways generally operative in or identical to those in animals and pathways involving psbA or phosphofructokinase and those encoded by the plastid genome. The limits of a xe2x80x9cpathwayxe2x80x9d are defined as they are generally known to those of skill in the art. Methods to detect plant counterparts in Apicomplexan include: a) immunoassays using antibodies directed to products and enzymes known in plants; b) hybridization assays using nucleotide probes that hybridize to specific sequences in plants; c) determining homologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or protein sequences; and/or d) substrate tests for specific enzymatic activity.
The xe2x80x9cplant-likexe2x80x9d pathways of the present invention are identified by:
a) identification of metabolic pathways characteristic of plants but not generally present in animals;
b) identification and characterization of Apicomplexan enzymes, nucleic acids and transit sequences as components similar or homologous to those in a);
c) identification and development of compounds (inhibitors) which abrogate the effect of the components of the pathways in vitro and in vivo, singly or in a plurality, against one or more types of Apicomplexan parasites and in conjoint Apicomplexan, bacterial and fungal infections.
The identified pathways are then used for:
a) rational design or selection of compounds more active than the known compounds (inhibitors), with good absorption following oral administration, with appropriate tissue distribution and without toxicity or carcinogenicity;
b) testing of such rationally designed compounds alone and together for safety, efficacy and appropriate absorption and tissue distribution in vitro and in vivo;
c) development and testing of diagnostic reagents and assays;
d) development and testing of live attenuated and component based vaccines.
By locating new targets in Apicomplexan pathways, doors are now open for development of more effective antimicrobial agents to treat Apicomplexan parasites in humans and agricultural animals. In addition, enzymes in these plant-like pathways provide improved diagnostic tests for diseases caused by Apicomplexans. Vaccines against infectious diseases caused by Apicomplexan parasites are derived from the novel compositions of the invention.
A method for inhibiting an Apicomplexan parasite, includes selecting the metabolic pathway of the present invention and interfering with the operation of the pathway in the parasite. The Apicomplexan parasite is preferably selected from the group that includes Toxoplasma, Plasmodium, Cryptosporidia, Eimeria, Babesia and Theileria. The pathway may utilize a component encoded by an Apicomplexan nuclear gene.
Suitable metabolic pathways or components include:
a) synthesis of heme from glutamate and tRNA glu by the plant-like, heme synthesis (5 carbon) pathway (hereinafter the xe2x80x9cheme synthesis pathwayxe2x80x9d);
b) synthesis of C4 acids (succinate) by the breakdown of lipids into fatty acids and then acetyl CoA, and their use in the glyoxylate cycle (hereinafter the xe2x80x9cglyoxylate cyclexe2x80x9d);
c) synthesis of chorismate from phosphoenolpyruvate and erythrose 4 phosphate by the shikimate pathway (hereinafter the xe2x80x9cshikimate pathwayxe2x80x9d);
d) synthesis of tetrahydrofolate from chorismate by the shikimate pathway;
e) synthesis of ubiquinone from chorismate by the shikimate pathway;
f) electron transport through the alternative pathway with use of the alternative oxidase (hereinafter the xe2x80x9calternative oxidase pathwayxe2x80x9d);
g) transport of proteins into or out of organelles through the use of transit sequences;
h) synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from chorismate by the shikimate pathway;
I) synthesis of the menaquinone, enterobactin and vitamin K1 from chorismate by the shikimate pathway;
j) synthesis of the branched chain amino acids (valine, leucine and isoleucine) from pyruvate and ketobutyrate by the plant-like branched chain amino acid synthesis pathway;
k) synthesis of the xe2x80x9cessentialxe2x80x9d (i.e., not synthesized by animals) amino acids, histidine, threonine, lysine and methionine by the use of plant-like amino acid synthases;
l) synthesis of linoleneic and linoleic acid;
m) synthesis of amylose and amylopectin with starch synthases and Q (branching) enzymes and their degradation;
n) synthesis of auxin growth regulators from indoleacetic acid derived from chorismate;
o) synthesis of isoprenoids (diterpenes, 5 carbon units with some properties of lipids) such as giberellins and abscidic acid by the mevalonic acid to giberellin pathway.
The interfering compositions are selected from the group consisting of enzyme inhibitors including competitors; inhibitors and competitive or toxic analogues of substrates, transition state analogues, and products; antibodies to components of the pathways; toxin conjugated antibodies or components of the pathways; antisense molecules; and inhibitors of transit peptides in an enzyme. In particular, the interfering compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG. Interfering with the operation of the metabolic pathway is also accomplished by introducing a plurality of compositions to the pathway, wherein each of the compositions singly interferes with the operation of the metabolic pathway. In certain instances, the plurality of compositions inhibits the parasite to a degree greater than the sum of the compositions used singly, that is exhibits a synergistic effect. Embodiments of a plurality of compositions include gabaculine and sulfadiazine; NPMG and sulfadiazine; SHAM and gabaculine, NPMG and pyrimethamine; NPMG and cycloguanil (which inhibits Apicomplexan DHFR[TS]), and other inhibitors and competitors of interrelated cascades of plant-like enzymes. Wherein the effect of inhibitors together is greater than the sum of the effect of each alone, the synergistic combination retards the selection of emergence of resistant organisms and is more effective than the individual components alone.
In various embodiments, the interfering composition acts on a latent bradyzoite form of the parasite, or multiple infecting Apicomplexan parasites simultaneously, or on conjoint infections with other pathogenic microorganisms which also utilize the plant-like metabolic pathway.
A method of determining the effectiveness of a composition in reducing the deleterious effects of an Apicomplexan in an animal, include: a) identifying a composition that inhibits growth or survival of an Apicomplexan parasite in vitro by interfering with a plant-like metabolic pathway and b) determining a concentration of the composition in an animal model that is non-toxic and effective in reducing the survival of the parasite in the animal host and/or the deleterious effects of the parasite in the animal.
Developing a lead compound that inhibits an Apicomplexan parasite is accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan parasite and b) identifying a composition that interferes with the operation of the pathway as a lead compound.
A composition which inhibits a specific life cycle stage of an Apicomplexan parasite by interfering with a plant-like metabolic pathway that utilizes a component encoded by a nuclear gene includes gabaculine; a composition including an enzyme in a metabolic pathway in an Apicomplexan parasite that is selectively operative in a life cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycosyl transferase. A composition comprising SHAM and 8-OH-quinoline inhibits the alternative oxidase in the latent bradyzoite form of an Apicomplexan parasite.
A method to identify a plant-like gene encoding a component of a plant-like metabolic pathway in an Apicomplexan parasite is a) obtaining a strain of E. coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain to require supplemented media for growth, b) complementing the E. coli with a gene or portion of the gene encoding a component of the metabolic pathway in the Apicomplexan parasite; and c) determining whether the complemented E. coli is able to grow in unsupplemented media, to identify the gene.
Another method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite with a gene probe; and b) determining whether the probe has complexed with the parasite from which the identity of the gene product is inferred.
A method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite also includes: a) cloning and sequencing the gene; and b) determining whether the gene is homologous to a plant gene which encodes a plant enzyme with the same function.
A method for identifying a plant-like gene product in a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for the plant-like enzyme; b) measuring enzyme activity; c) determining whether the enzyme is operative; and d) inhibiting activity of the enzyme in vitro with an inhibitor.
Identifying a gene or gene product in an Apicomplexan parasite which possesses an organelle transit sequence which transports a protein, wherein the protein is not necessarily an enzyme in a metabolic pathway, but is identified because it has a characteristic organelle transit sequence is also within the scope of the invention.
The invention also relates to a diagnostic reagent for identifying the presence of an Apicomplexan parasite in a subject, where the subject includes a domestic or livestock animal or a human. The reagent may include all or a portion of a component of the plant-like pathway, an antibody specific for an enzyme that is a component of a plant-like metabolic pathway in the parasite, or all or part of a nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathway. A diagnostic assay that identifies the presence of an Apicomplexan parasite or specific life-cycle stage of the parasite may use the diagnostic reagents defined herein.
A diagnostic reagent for identifying the presence of an Apicomplexan parasite, includes an antibody specific for an enzyme that is part of a plant-like metabolic pathway.
A diagnostic assay for the presence of an Apicomplexan parasite in a biological sample includes: a) contacting the sample with an antibody selective for a product of a plant-like metabolic pathway that operates in an Apicomplexan parasite; and b) determining whether the antibody has complexed with the sample, from which the presence of the parasite is inferred. Alternatively, the assay is directed towards a nucleotide sequence. In both these cases, appropriate antibody or nucleotide sequences are selected to distinguish infections by different Apicomplexans.
An aspect of the invention is a vaccine for protecting livestock animals, domestic animals or a human against infection or adverse consequences of infection by an Apicomplexan parasite. The vaccine may be produced for an Apicomplexan parasite in which a gene encoding a component of a plant-like metabolic pathway in the parasite is manipulated, for example, deleted or modified. When the gene is deleted or modified in the live vaccine, the component of the pathway may be replaced by the presence of the product of an enzymatic reaction in tissue culture medium. The vaccine strain can then be cultivated in vitro to make the vaccine.
A vaccine for protecting animals against infection by an Apicomplexan parasite is based on an Apicomplexan parasite in which the parasite or a component of a metabolic pathway in the parasite is used.
The vaccine may use a component of the pathway that is operative at a particular life stage of the parasite. A suitable component is the AroC gene from T. gondii or P. falciparum. 
A method of treatment for an infection in a subject by an Apicomplexan parasite includes the following steps: a) obtaining an inhibitor of a plant-like metabolic pathway in an Apicomplexan parasite; and b) administering an effective amount of the inhibitor to the subject.